Method, reagents and kit for diagnosis and targeted screening for p53 mutations

ABSTRACT

Rapid and cost effective diagnosis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost: 
     (a) immunoassays, 
     (b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene. Preferably, the amplification primers are multiplexed so that more than one DNA fragment is amplified in a single vessel, using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal p53 gene; and 
     (c) analysis of DNA from a patient sample by DNA sequencing of the p53 gene beginning with the sequencing of those regions most likely to harbor point mutations, and proceeding to sequence regions less likely to harbor point mutations.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/271,946 filed Jul. 8, 1994, now U.S. Pat. No. 5,545,527 anda continuation-in-part of U.S. patent application Ser. No. 08/388,381filed Feb. 14, 1995, now U.S. Pat. No. 5,552,283.

BACKGROUND OF THE INVENTION

This application relates to a method, reagents and kit for diagnosis andtargeted screening of mutation in p53 protein and mutation in the genecoding for the p53 protein (herein the "p53 gene") Such mutations arecollectively referred to herein as "p53 mutations".

The evidence for the tumor suppressor activity of wild-type p53 is nowextensive. Since its discovery in 1979 when it was found to be complexedwith the SV40 large T antigen in SV40-transformed rodent cells thesignificance of p53 has slowly come to light. The protein appears to actas a transcription factor, and may be responsible for apoptosis ofpre-cancerous cells. (Ziegler et al., "Sunburn and p53 in the onset ofskin cancer", Nature 372: 773-776 (1994)) The DNA sequence of the 11exons of the p53 gene is now known and close to 1000 papers werepublished on p53 in 1993.

P53 mutations are significant because they are found in an enormousvariety of tumors. Among common tumors, about 70% of colorectal cancers,50% of lung cancers, and up to 40% of breast cancers carry p53 genemutations. p53 is also linked to cancers of the blood and lymph nodes,including Hodgkin's disease, T cell lymphoma and certain kinds ofleukemia. Moreover, aberrant forms of the p53 gene are correlated withmore aggressive tumors, metastasis and lower 5-year survival rates. Suchreports have emerged for cancers of the colon, lung, cervix, bladder,prostate, breast and skin.

The serious consequences of p53 mutations mandates a method fordetection and diagnosis of such mutations which is rapid and accurateand that can be performed at the earliest stage of tumor development.Toward this end, immunoassays and DNA assays for p53 mutations are knownin the art. To date, however, neither method has been able to identifyp53 mutations with a high degree of specificity and accuracy in a costeffective fashion. Those immunoassays that have been published identifyonly a small portion of those patients actually thought to be carryingthe mutation.

Two general techniques of immunoassay have been employed. The firsttechnique is an indirect method which detects anti-p53 antibodies thatarise in some patients who have p53 mutations. Immunoassay tests fordetecting anti-p53 antibodies in patient sera are currently based onradio-active labeling, immunoprecipitation and immunoblotting. All thesemethods are qualitative and time consuming and thus not suitable forscreening large number of samples. (Angelopoulou et al., "Autoantibodiesagainst the p53 tumor suppressor gene product quantified in cancerpatient serum with time-resolved immunofluorometry", Cancer J 6(6):315-321 (1993)).

The second technique of immunoassay directly detects mutant p53 protein.Such methods have been disclosed in at least two publications. Bartek etal. disclosed an enzyme-linked immunosorbent assay for p53 which wasapplied for the measurement of p53 in tumor tissue extracts (Oncogene,6: 1699-1703 (1991)). Hassapoglidou et al. developed a monoclonalantibody useful for direct detection of mutant p53. (Oncogene 8:1501-1509 (1993).)

DNA analysis of p53 mutations has been reported using at least twotechniques. First, single stranded conformational polymorphism was usedto detect mutant p53 by Kuypers et al. ("Detection of point mutations inDNA using capillary electrophoresis in a polymer network", J.Chromatography 621: 149-56 (1993)) and by Felix et al ("Absence ofhereditary p53 mutations in 10 familial leukemia pedigrees", J ClinInvest 90: 653-8 (1992)). Second, actual genomic DNA sequencingdiagnosis was performed on relatively small groups of patients byToguchida et al. ("Prevalence and spectrum of germline mutations of thep53 gene among patients with sarcoma", N Engl J Med 326: 1301-8 (1992))and by Malkin et al. ("Germline mutations of the p53 tumor-suppressorgene in children and young adults with second malignant neoplasms", NEngl J Med 326: 1309-15 (1992)). The cDNA sequence of a larger group ofpatients (over 400) was reported in a Pharmacia LKB meeting publication(Andell et al. "A new approach in automated DNA sequencing to analysethe p53 gene in a large number of breast cancer patients", Pharmacia LKBmeeting literature, 1994) These rapid DNA-based techniques have beenused to detect mutations, but because they are so labor intensive, thatlarge-scale screening tests are impractical (Harris et al., "Clinicalimplications of the p53 tumor-suppressor gene", N Engl J Med329:1318-1327 (1993)).

Thus, the existing methods of diagnosis have been frustratinglyunsatisfactory. Researchers have used either immunoassay or DNAanalytical methods to diagnose p53 mutation, even though such testsresult in numerous false negatives. A method is required for rapid andcost effective diagnosis of p53 mutation in the millions of individualswho develop potentially life threatening malignancies each year.

It is an object of this invention to provide a method for rapid and costeffective diagnosis of p53 mutations in a sample of patients.

It is a further object of this invention to provide DNA sequencing andamplification primers specific for analysis of the p53 gene from apatient sample.

It is a further object of this invention to provide kits of DNAoligonucleotides for amplification and sequencing of the p53 gene of apatient.

It is a further object of this invention to provide a method ofgenerating a p53 mutation report which is used to provide appropriategenetic counseling to the patient and family upon whom the test isperformed.

SUMMARY OF THE INVENTION

In accordance with the present invention, rapid and cost effectivediagnosis of p53 mutations of a sample of patients is achieved byemploying a selected plurality of diagnostic tools, in a hierarchy ofincreasing accuracy and cost per tool, in which each tool detectsessentially no false positives. Diagnostic tests that may be includedamong the plurality of tests selected include, in order of increasingaccuracy and cost:

(a) immunoassays, particularly immunoassays for anti-p53 antibodiespresent in a patient sample;

(b) analysis of DNA from a patient sample by quantitative amplificationof p53 exons using amplification primers complementary to intron regionsflanking each exon and examination of the length or quantity of eachamplified fragment for nucleotide insertions or deletions relative tothe normal p53 gene. Preferably, the amplification primers aremultiplexed so that more than one DNA fragment is amplified in a singlevessel, using sets of primers which provide gene fragments ofdistinctive lengths when used to amplify a normal p53 gene; and

(c) analysis of DNA from a patient sample by DNA sequencing of the p53gene beginning with the sequencing of those regions most likely toharbor point mutations, and proceeding to sequence regions less likelyto harbor point mutations.

The present invention includes a multitude of amplification andsequencing primers. These primers, taken individually or as part of kitsfor the detection of p53 mutations represent a further aspect of thepresent invention. Particularly preferred primers constitute sets thatare compatible for coamplification and that produce amplified DNAfragments of distinctive lengths from other fragments amplified in thesame set.

The information obtained in the test is used to generate a report whichis used to provide appropriate genetic counseling to the patient andfamily upon whom the test is performed. The generation of such reports,which may be in the form of a printed report, an electroniccommunication, such as a facsimile or electronic mail (e-mail)transmission, or a posting of a data entry in a computer record relatingto the patient, is a further aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the hierarchy of p53 diagnosisas provided by the invention; and

FIG. 2 outlines two methods of immunoassay for the presence of anti-p53antibodies in patient sera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the identification of p53mutations, a method for generating reports for providing counseling topatients and families of patients with p53 mutations, and tooligonucleotide primers and kits useful in practicing these methods. Thepresent invention utilizes the hierarchical approach which is disclosedgenerally in U.S. patent application Ser. No. 08/271,946, and in aconcurrently filed continuation-in-part thereof, PCT/US95/08606, whichis incorporated herein by reference.

The method for identification of p53 mutations in a sample of patientsis based upon using diagnostic tools 1) in a hierarchy of increasingaccuracy and cost per tool; and 2) in which each tool is of extremelyhigh specificity and detects essentially no false positives. A samplewhich exhibits a positive result establishes the presence of a p53mutation and a patient report can be prepared on this basis. A samplewhich exhibits a negative result is thereafter subjected to a morecostly, but more accurate test to determine if a mutation is present.

Thus, as shown in FIG. 1, an example of such a hierarchy comprises, inorder, an immunoassay step; a DNA fragment length/quantity analysis; andDNA sequencing. In the exemplary results shown in FIG. 1, the first stephas the advantage of low cost, but detects a positive result in only 15%of the patients. The second level of the test hierarchy is of moderatecost, but because of its increased accuracy it detects a positive resultin 30% (26 of 85) of the patients who tested negative using theimmunoassay. This means that the more expensive DNA Sequencing analysisneed only be performed on 59% of the original patients. Thus, the use ofthe hierarchical approach leads to a substantial reduction in theaverage cost of the test while providing high levels of accuracy forevery patient, by having eliminated from the test pool those samplesthat are known to be positive.

IMMUNOASSAY PROCEDURES

As illustrated in FIG. 1, the first level in the hierarchy of p53diagnosis may be an immunoassay. Because of the relatively low cost ofimmunoassay, it may be advantageously used to eliminate a significantnumber of patients from the sample pool prior to proceeding to the nextlevel of the hierarchy. Methods of immunoassay that can be employed atthis first stage have been discovered by many researchers. The followinglist of papers all include methods for identifying p53 mutations byimmunoassay, though by no means is this list exclusive, and apractitioner skilled in the art may know of alternative methods of p53immunoassay:

Angelopoulou et al., "Autoantibodies against the p53 tumor suppressorgene product quantified in cancer patient serum with time-resolvedimmunofluorometry", Cancer J 6(6): 315-321 (1993).

Bartek et al., Oncogene 6: 1699-1703 (1991).

Crawford et al., "Detection of antibodies against the cellular proteinp53 in sera from patients with breast cancer", Int J Cancer 30: 403-8(1982).

Caron De Froentel et al., "Presence of circulating antibodies againstcellular protein p53 in a notable proportion of children with B-celllymphoma", Int J Cancer 39: 185-9 (1987).

Christopoulos et al., "Oncogenes and tumor suppressor genes: newbiochemical tests." CRC Crit Rev Clin Lab Sci 29: 269-305 (1992).

Davidoff et al., "Immune response to p53 is dependent upon p53 HSP70complexes in breast cancers", Proc Natl Acad Sci USA 89: 3439-42 (1992).

Hassapoglidou et al., "Antibodies to the p53 tumor suppressor geneproduct quantified in cancer patient serum with a time-resolvedimmunofluorometric technique", Clin Biochem 25:445-9 (1992).

Hassapoglidou et al., "Quantification of p53 protein in tumor celllines, breast tissue extracts and serum with time-resolvedimmunofluorometry", Oncogene 8:1501-1509 (1993).

Labrecque et al., "Analysis of the anti-p53 antibody response in cancerpatients", Cancer Res 53: 3468-71 (1993).

Schlichtholz et al., "The immune response to p53 in breast cancerpatients is directed against immunodominant epitopes unrelated to themutational hot spot", Cancer Res 52: 6380-84 (1992).

Volkmann et al., "The humoral immune response to p53 in patients withhepatocellular carcinoma is specific for malignancy and independent ofthe alpha-fetoprotein status", Hepatology 18: 559-565 (1993).

Winter et al., "Development of antibodies against p53 in lung cancerpatients appears to be dependent on the p53 mutation", Cancer Res 52:4168-74 (1992).

In selecting an immunoassay for use in the present method, it should beunderstood that some immunoassays only detect small subsets of p53mutations, while other immunoassays can detect a wide variety of p53mutations. In order to reduce the cost of diagnosis, it is advantageousto take into account the scope of detection of each immunoassay andselect an immunoassay which provides highly specific detection for thegreatest number of p53 mutations as the analytical start point. Ifdesired, a second immunoassay may be used after the first immunoassay ifit will identify other types of mutations not found by the firstimmunoassay. This selection and sequential ordering of immunoassays maycontinue until the patient pool is reduced to eliminate essentially allpatients reasonably diagnosed with immunoassay techniques before goingon to other, more expensive and more accurate assays.

DNA ANALYSIS

The next level in the hierarchy of this invention test those patientsamples which did not prove positive for p53 mutation based onimmunoassay, be subjected to more accurate and more costly analysis. DNAanalysis of patient samples is an non-limiting example of such ananalysis. Within the general class of DNA analysis, however, there is ahierarchy of methods, each level in the hierarchy providing increasingaccuracy but at increased cost. At the top end of this hierarchy (mostaccurate and expensive) is DNA sequencing. DNA sequencing provides themaximum degree of accuracy because each base in DNA from the sample isidentified and compared to the wild-type sequence.

DNA analysis also provides the option of methods which are lower in thehierarchy than DNA sequencing but which offer greater accuracy thanimmunoassay procedures. A non-limiting example of such a test is anassay for DNA fragment length/quantity mutations. This fragment analysiscan be used as an intermediate level in a three (or more) levelhierarchy, or it can be used as the first level of a hierarchy in placeof the immunoassay tests described above.

FRAGMENT ANALYSIS

As illustrated in FIG. 1, level two of the hierarchy of this inventionmay be DNA fragment length/quantity analysis. In this test, one or moreexons of the p53 gene are quantitatively amplified using primersdesigned to create amplification products of known length and thelengths and/or quantity of the amplified fragments are determined. Ifthere is a variance between the length of any amplified exon, and thenormal length of that exon, this is an indication of an insertion ordeletion mutation in that exon. The quantity of amplified material fromamplification of a sample exon may also reflect the loss of geneticmaterial. In particular, by comparing the quantity of amplifiedmaterials produced to standards amplified from a null allele (0 copiesof p53), a hemizygous standard (1 copy of p53), a wild type standard (2copies of p53) and a trisomy standard (3 copies of p53), the nature ofthe mutation may be further investigated.

The number of exons tested in step two of the hierarchy is a matter ofchoice for the user. For example, if after repeated testing on patientsamples it is found that length or quantity mutations are rarely foundin certain exons, it is preferable to test these exons last, aftertesting other exons to see if a mutation sufficient to cause the diseaseis detected, before incurring the expense to test these less likelyexons. In testing these other exons, the user may choose to test themone at a time, or in one multiplexing group at a time. Alternatively,the user may choose to test all exons simultaneously at once.

When a length mutation is detected in this step of the hierarchy, it isnot necessary to perform additional tests on the patient sample tocomplete the identification process. Preferably, however, the sequenceof the mutated exon will be determined as part of the second level ofthe hierarchy to confirm that the mutation detected can in fact be acause of the observed disease.

Again, all those samples that prove positive for a length or quantitymutation are eliminated from the patient sample, and patient reports maybe made for each patient eliminated. However, if no mutation is detectedat this level of the hierarchy, then a more accurate and costly analysismust be undertaken. DNA sequence analysis is a non-limiting example ofsuch a level.

The primers used to amplify the sample DNA for fragment analysis areoligonucleotides of defined sequence selected to hybridize selectivelywith particular portions of the p53 gene, generally introns. Each primerhas bound to it a detectable label. A preferred example of such a labelis fluorescein, which is a standard label used in nucleic acidsequencing systems using laser light as a detection system. Otherdetectable labels can also be employed, however, including otherfluorophores, radio-labels, chemical couplers such as biotin which canbe detected with streptavidin-linked enzymes, and epitope tags such asdigoxigenin detected using antibodies available fromBoehringer-Mannheim.

While considerable variation is possible in the sequence of the primersused in amplifying the exons as part of the method of the presentinvention, the primers used in amplification and the conditions of theamplification are preferably optimized for use in the present invention.Looking first at the primers used, it will be understood that in orderto avoid the possibility of false positive results the primer pair,i.e., the combination of the 5'-primer and the 3'-primer for any givenexon must be unique to the p53 so that only the p53 gene will beamplified. This means that the primer sequences will be generallysomewhat longer than the minimum which can be used as an amplificationprimer. Preferred primers are from 18 to 23 nucleotides in length,without internal homology or primer--primer homology. It is alsodesirable for the primers to form more stable duplexes with the targetDNA at the primers' 5'-ends than at their 3'-ends, because this leads toless false priming. Stability can be approximated by GC content, sinceGC base pairs are more stable than AT pairs, or by nearest neighborthermodynamic parameters. Breslauer et al., "Predicting DNA duplexstability from base sequence", Proc. Nat'l Acad. Sci. USA 83: 3746-3750(1986). In addition, to ensure complete amplification of each exon, thetwo primers of a pair are preferably selected to hybridize in theintrons immediately flanking the exon to be amplified using the primerpair.

Additional factors apply to the selection of primers for multiplexedamplification of exons, i.e., where several exons are amplifiedconcurrently in a single reaction mixture. These factors are discussedin Rylchik, W., "Selection of Primers for Polymerase Chain Reaction", inMethods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White, B. A. ed., Humana Press, Totowa, N.J., 1993.Briefly, applying these factors, primer pairs are selected by position,similarity of melting temperature, internal stability, absence ofinternal homology or homology to each other, i.e., they won't stick toeach other or to themselves, and the 3'-end will not form a stablehairpin loop back on itself.

Thus, in the present case, the goal is to have sets of primer pairs withapproximately the same thermal profile, so that they can be effectivelycoamplified together. This goal can be achieved by having groups ofprimer pairs with approximately the same length and the same G/Ccontent. In addition, it is preferred that the length of the gene regionbetween the primer binding sites on a normal p53 gene differ for eachexon to be multiplexed as a group. Differences of only one base inlength are sufficient, provided a high resolution gel capable ofresolving one base differences is used in analyzing the amplificationproducts. However, greater differences in length are preferred.

To evaluate compatibility of primers for use in coamplification, it isdesirable to determine the predicted melting temperature for eachprimer. This can be accomplished in several ways. For example, themelting temperature, Tm can be calculated using either of the followingequations:

    Tm (° C.)=81.5+16.6×log [Na]+0.41×(%GC)-675/length

where [Na] is the concentration of sodium ions, and the %GC is in numberpercent, or

    Tm (° C.)=2×(A+T)+4×(G+C)

where A, T, G, and C represent the number of adenosine, thymidine,guanosine and cytosine residues in the primer. In general, primers forcoamplification should be selected to have predicted meltingtemperatures differing by less than 4° C.

DNA SEQUENCE ANALYSIS

FIG. 1 illustrates that the final element of the hierarchy of p53diagnosis is DNA sequencing. DNA sequence analysis involves determiningthe sequence of the exons to locate the mutation.

Sequencing is expensive and so it may be desirable to use asub-hierarchy within this level of testing to reduce the likelihood ofhaving to sequence all of the exons. In this case a suitablesub-hierarchy will be determined by identifying those exons whereinmutations are most likely to occur. Mutational hotspots have beenidentified at codons 175, 245, 248, 249, 273 and 282, which correspondto exon 5 for the first listed hotspot, exon 6 for the second threelisted hotspots and exon 7 for the latter two hotspots. (Harris CC. p53:At the Crossroads of Molecular Carcino-genesis and Risk Assessment.Science 1993; 262: 1980-1981.) In accordance with this sub-hierarchy,the first exons sequenced are those which are easy to sequence and whichcontain hotspots. Next, if no mutation is found, hotspot exons aresequenced which are hard to sequence (i.e. are found empirically to giveless clear results when treated similarly to other exons). Finally, theremaining exons are sequenced in descending order of the odds of findinga mutation based on prior epidemiological studies. (The order ofsequencing of the exons may change as patient data accumulates on thelocation of point mutation hotspots.) If no mutation has been detectedafter all the exons have been sequenced, then it is concluded that thereis no mutation in the test sample and a report is generated accordingly.

A preferred method for DNA sequencing as part of the method of theinvention involves amplifying the exon of interest and then determiningits sequence. Amplification of each exon may be performed using the sameprimers employed for fragment length analysis, although the detectablelabel included for the fragment analysis is not necessary foramplification prior to sequencing. In this case, however, multiplexedamplification cannot be used since only a single amplification productis desired.

The determination of the sequence of the amplified material may becarried out in any manner. A preferred approach, however, is thewell-known Sanger method involving a template-dependent primer extensionreaction in the presence of dideoxy chain terminating nucleotides. Forthis method, a sequencing primer is used which hybridizes to one chainof the amplified DNA. In general, sequencing primers are nested insidethe amplification primers, although the amplification primers could beused for sequencing purposes if desired.

The amplification and sequencing primers used in the present inventionare advantageously packaged as kits for the detection of mutations inthe p53 gene. The primers may be packaged individually within the kit,or as mixtures of primers, sometimes referred to as "primer cocktails,"which are useful in a single reaction vessel. Such kits may contain asingle pair of primers, useful for quantitative amplification of asingle exon, or multiple pairs of primers useful for amplification ofmultiple exons. Such kits may further include amplification and/orsequencing primers for one or more exons. Such kits may also includereagents other than primers for use in the amplification reaction, suchas a polymerase and buffers, but this is optional.

Preferred kits in accordance with the invention comprise a plurality ofprimer pairs useful in the co-amplification of a plurality of exons ofthe p53 gene. Primer pairs in such kits are selected to have a commonmelting temperature and to produce amplification products havingdiffering lengths.

The following non-limiting examples illustrate applications of theinvention.

EXAMPLE 1

In order to determine the presence or absence of p53 mutations in asample of patients, the following hierarchy of tests was performed.

Level 1 Immunoassay: Detection of anti-p53 antibodies

The two methods of Angelopoulou and Diamandis (1993, supra) wereemployed to detect anti-p53 antibodies in a patient sample. FIG. 2 Aillustrates the principle of Method A for anti-p53 antibodyquantification in patient sera. In the presence of such antibodies, a"sandwich" is formed between a mouse monoclonal anti-p53 antibody andhuman anti-p53 antibodies, resulting in high fluorescence readings. FIG.2 B illustrates the principle of Method B for p53 antibodyquantification in patient sera. The left-hand panel shows that in theabsence of anti-p53 antibodies in human serum, the added p53 antigen ismeasured as shown, giving rise to fluorescence. The right-hand panelshows that when anti-p53 antibodies are present in patient serum, theyblock the added p53 antigen, resulting in low fluorescence. No sandwichcan be formed between the mouse monoclonal and rabbit polyclonalanti-p53 antibodies. ALP=alkaline phosphatase; FSAP=fluorosalicylphosphate; FSA=fluorosalicylate; Ab=antibody.

Instrumentation and Materials--For measuring liquid-phase Tb3+fluorescence in white microtiter wells, a Cyberfluor 615 Immunoanalyzertime resolved fluorometer was used as described elsewhere(Christopoulous et al., supra; Papanastasiou-Diamandi et al.,"Ultrasensitive thyrotropin immunoassay based on enzymatically amplifiedtime-resolved fluorescence with a terbium chelate", Clin Chem 38:545-48(1992)). The phosphate ester of 5-fluorosalicylic acid (FSAP) wasobtained from CyberFluor Inc., Toronto, Canada. TbCl3. 6H20 was from GfSChemicals, Columbus Ohio, USA. All other chemicals were form SigmaChemical Co., St. Louis, Mo., USA unless otherwise stated.

Solutions--The enzyme substrate buffer was a 0.1 mol/L Tris solution, pH9.1, containing 0.1 mol NaCl and 1 mmol MgCl₂ per liter. The stock FSAPsubstrate solution was a 10⁻² mol/L solution in 0.1 mol/L NaOH. FreshFSAP substrate working solutions were prepared just before use bydilution (10-fold) of the stock in the enzyme substrate buffer. The celllysis buffer was a 20 mmol/L Tris solution, pH 8.1, containing 150 mmolNaCl, 10 g Nonidet P-40, 0.5 mmol phenylmethylsulfonyl fluoride, 2 mgleupeptin and 2 mg aprotinin per liter. The developing solution was a 1mol/L Tris base solution containing 0.4 mol NaOH, 3 mmol EDTA and 2 mmolTbCl₃.6H₂ O per liter (no pH adjustment). The washing solution was a 5mmol/L Tris buffer, pH 7.80, containing 0.5 g Tween 20 and 150 mmol NaClper liter. The coating antibody solution was a 50 mmol/L Tris buffer, pH7.80, containing 0.5 g sodium azide per liter. The CM-1 antibody diluentwas a 50 mmol/l Tris buffer, pH 7.80. containing 60 g bovine serumalbumin (BSA) per liter. The goat anti-rabbit immunoglobulin alkalinephosphatase conjugate (GARRIg-ALP) diluent was a 50 mmol/l Tris buffer,pH 7.80, containing 60g BSA, 0.5 mol KCl and 100 ml goat serum perliter.

The cell lines used in this study were colon carcinoma Colo 320 HSR(+)(Murakami et al., "Detection of aberrations of the p53 alleles and thegene transcript in human tumor cell lines by single-strand conformationpolymorphism analysis", Cancer Res 51: 3356-61 (1991)); pancreaticcarcinoma MIA PaCa-2 (Barton et al. "Abnormalities of the p53 tumorsuppressor gene in human pancreatic cancer", Br J Cancer 64:1076-821991)); breast carcinoma T-47D (Bartek et al., "Genetic andimmunochemical analysis of mutant p53 in human breast cancer celllines", Oncogene 5:893-9 (1990)); and human erythroleukemia OCI M2(Singerland et al., "Mutations of the p53 gene in human acutemyelogenous leukemia", Blood 77: 1500-7 (1991)).

These cell lines were cultured as described elsewhere; they all have p53gene mutations and overproduce mutant p53 protein (Hassapoglidou et al.,1993, supra). Recombinant wild type p53 protein, produced as describedelsewhere (Wang et al., "The murine p53 blocks replication of SV40 DNAin vitro by inhibiting the initiation functions of SV40 large Tantigen", Cell 57: 379-92 (1989)), was a gift by Dr. Carol Prives,Columbia University, N.Y.

Lysates from cell lines producing p53, or recombinant p53 were dilutedin a 50 mmol/L Tris buffer, pH 7.80, containing 60 g of BSA per literfor Method A and in 100% goat serum for Method B. The mouse monoclonalanti-p53 capture antibody (Pab240) diluent was a 50 mmol/L Tris buffer,pH 7.80, containing 60 g BSA and 0.5 mol KCl per liter. Serum sampleswere diluted in a serum diluent which is the same as the Pab240 diluentbut supplemented with 10% normal goat serum and 2% normal mouse serum.The goat anti-human immunoglobulin-alkaline phosphatase conjugate(GAHIg-AALP) diluent was the same as the GARIg-ALP diluent.

The mouse anti-p53 monoclonal antibody Pab240 was produced as a tissueculture supernatant from a cell line donated to us by Dr. D. P. Lane,University of Dundee, U.K. Its antibody concentration was approximately30 micrograms/ml. The rabbit polyclonal anti-p53 antibody, CM-1, wasobtained from Dimension Labs, Mississauga Ontario. The goat anti-rabbitand goat anti-human antibodies, conjugated to alkaline phosphatase, andthe goat anti-mouse antibody, Fc fragment specific (GAMIg), allapproximately 1 mg/ml, were obtained from Jackson Imunoresearch, WestGrove, Pa.

Patient Sera--Sera from cancer patients were stored at -70° C. untilanalysis. Sera used were from patients with breast (n=105), ovarian(n=72), colon (n=77) and pancreatic cancer (n=46). For correlationstudies 38 p53 antibody-positive sera from patients with the abovemalignancies was used plus sera from prostate, lymphoma, lung andmultiple myeloma patients.

Procedures

Cell Lysis--Cells from each cell line were grown until they reachedapproximately 10×6 cells/ml or 90% confluency. The cell pellet from a 15ml culture was lysed in 300 microliters lysis buffer, for 30 min, onice. The cell extract was centrifuged at 12,000×g for 10 min and thepellet discarded. The lysate was used within two hours. Total proteinwas measured in the lysates with the bicinchoninic acid (BCA) assay,commercially available from Pierce Chemical Co., Rockford Ill. Lysatestypically contained 1-3 mg of protein per ml.

Immunoassay Procedure, Method A--This method is a modification of anassay previously published (Hassapoglidou 1992, supra). White, opaque,12-well microtiter strips (from Dynatech laboratories, Alexandria, Va.)were coated with goat anti-mouse immunoglobulin diluted 500-fold in thecoating antibody diluent (100 microliters/ 200 ng/well, overnightincubation at room temperature). This indirect coating is superior todirect coating with the Pab240 antibody. The wells were then washed sixtimes with an automatic washer and used for the assay as follows: 50microliters of cell lysate (diluted 10-fold in the cell lysate diluent)and 100 microliters of mouse monoclonal anti-p53 antibody Pab240(diluted 20-fold in the PAb240 diluent) were added and incubated for 3 hwith shaking at 37 C (air oven). After 6 washes, 100 microliter/well ofserum sample (diluted 10-fold in the serum sample diluent, in duplicate)and incubate for 1 h with shaking, at room temperature. After sixwashes, 100 microliters/well of an alkaline phosphatase-labeled goatanti-human immunoglobulin G-antibody (diluted 15,000 fold in theGAHIg-ALP diluent) was added. The wells were incubated for 1 h withshaking at room temperature and washed six times. 100 microliters/wellof the diluted FSAP substrate solution was added and incubated for 10min with shaking at room temperature. 100 microliters/well of thedeveloping solution, was added, mixed for 1 min and the fluorescence wasmeasured on a Cyberfluor 615TM immunoanalyzer.

Each assay run was accompanied by a parallel run to assess anynonspecific binding effects. This run was identical to the proceduredescribed above but the cell lysate was replaced by the lysate diluent.Sera were considered positive for antibodies only if the signal with thelysate exceed the signal without the lysate by a factor of 1.7(Hassapoglidou 1992, supra).

Immunoassay Procedure Method B--Microtiter strips were coated as inMethod A. Patient serum (200 microliters) was then incubated in tubeswith 20 microliters of a 10-fold diluted cell lysate from Colo 320 HSR(+) cells, for 30 min at room temperature. The p53-supplemented sera (50microliters, in duplicate) were then added to goat anti-mouse IgG-coatedwells along with 100 microliters/well of mouse monoclonal anti-p53antibody Pab240, diluted 20 fold as in Method A. The wells wereincubated at 37 C for 3 h with shaking and washed six times. 100microliters/well of the rabbit anti-p53 polyclonal antibody (CM-1)diluted 5,000 fold in the CM-1 antibody diluent was added and incubatedfor 1 h at room temperature with shaking. After washing six times 100microliters/well of the alkaline phosphatase-labeled goat anti-rabbitimmunoglobulin (GARIg-ALP) diluted 5000-fold in the GARIg-ALPP antibodydiluent was added and incubated for 1 h at room temperature withshaking. The wells were washed six times and the procedure continued asin Method A from the point of adding the FSAP substrate solution. Eachserum sample was also assayed without the addition of the Colo 320HSR(+) cell lysate to assess the background signal. Sera were consideredpositive for antibodies only if the fluorescence signal in the presenceof serum was less than 50% of the fluorescence signal obtained with a 6%BSA solution as sample.

Quantification--Due to the lack of a suitable standard solution, anarbitrary system to calibrate Methods A and B was devised. Among thehighly p53 antibody-positive sera one was selected and arbitrarilydefined to have a concentration of 20,480 Units/L. This serum sample asthen used in dilutions to construct calibration curves for assays A andB from which the concentration of the other samples was calculated.

The results of the test identified the presence of anti-p53 antibodiesin 15-16% of ovarian and colon cancer patients. Antibody prevalence wasbetween 5-8% in patients with lung and breast tumors. There was arelatively low prevalence of detectable anti-p53 antibodies (3-4%) inpatients with pancreatic and prostate cancer and in patients withmultiple myeloma or lymphoma. In patients with other malignancies(hepatoma, melanoma, leukemia, Kaposi's sarcoma and testicularcarcinoma) the p53-antibody prevalence was similar to that of non-cancerpatients (less than 2%).

Patient reports were prepared for those samples which demonstratedpositive results. All those test samples which were not positive for thep53 mutation in the protein immunoassay were then analyzed at the nextlevel in the hierarchy of the invention.

Level 2 DNA Fragment Length/Quantity Analysis

DNA is prepared from the patient sample using a Qiagen QIAamp Kitaccording to accompanying directions. Briefly, an aliquot of the bloodsample, or a lymphocyte--containing fraction thereof, is combined withProteinase K, mixed, and allowed to incubate to lyse the cells. Ethanolis added and the lysate is transferred to a QIAamp spin column fromwhich DNA is recovered after several washings.

Quantitative fragment length and amount analysis is performed to assayfor 1) the presence of insertion or deletion mutations; and 2) whetherthe patient is homozygous or heterozygous for the insertion or deletionmutation. To perform the analysis, the genomic DNA is amplified in threesets using multiplexing amplification primers. Each 50 microlitermultiplexed PCR reaction contains 0.5 micrograms genomic DNA, 150 ng oreach primer, 3.6 mM each dNTP, 42.5 micrograms Bovine Serum Albumin, 5units Taq polymerase in a buffer containing 10% DMSO, 16 mM (NH₄)2SO₄,6.7 mM MgCl₂, 6.8 micro Molar EDTA (pH 8.0) and 1 mM β-mercaptoethanol.The reaction mixture was initially incubated at 94 degrees C for 5minutes and then subjected to 30 cycles of PCR in a Perkin-Elmer/Cetusthermocycler as follows:

Denaturation: 94 degrees C, 30 seconds

Annealing: 60, 62 or 64 degrees C (depending on whether primer set A, B,or C is being amplified, respectively) for 50 secs.

Extension: 70 degrees C, 60 seconds; final extension at 72 degrees for 3minutes

The amplification of the eleven exons of the p53 gene is advantageouslycarried out in three multiplex pools. In multiplex pool A, exons 1, 3,4, 5, 6, 9, 10 and 11 are amplified (along with a control sequence). Themembers of this pool are selected because they all use a hybridizationtemperature of 60° C., and none of the expected fragment lengths willoverlap in an electrophoresis gel. One of each pair of primers islabeled at the 5 prime end with an identifiable marker such asfluorescein, rhodamine or cyanine. The primers are:

    ______________________________________                                        EXON 1                                                                        P53-5X1MP                                                                     CGGATTACTT GCCCTTACTT GTCA                                                                             [SEQ 1]                                              P53-3X1MP                                                                     CCCCAGCCCC AGCGATTTT     [SEQ 2]                                              EXON 3                                                                        P53-5X3,4P                                                                    CATGGGACTG ACTTTCTGCT    [SEQ 3]                                              P53-3X3MP                                                                     CCACGGCAAC GCCCACTGT     [SEQ 4]                                              EXON 4                                                                        P53-5X4MP                                                                     CTGCTCCTCT CACTGCTCTT TTCA                                                                             [SEQ 5]                                              P53-3X3,4P                                                                    AAAGAAATCC AGCCCGATAC CG [SEQ 6]                                              EXON 5                                                                        P53-5X5, 6P                                                                   TGTTCACTTC TCCCCTGACT    [SEQ 7]                                              P53-3X5MP                                                                     CACCCCTCTC CTCTCTCCAC    [SEQ 8]                                              EXON 6                                                                        P53-5X6MP                                                                     CTGGCGCTGG AGAGACGACA    [SEQ 9]                                              P53-3X5,6P                                                                    GCAGCCCCAC TGACAACCA     [SEQ 10]                                             EXON 9                                                                        P53-5X9P                                                                      GCGGTCGAGC AGACCAACG     [SEQ 11]                                             P53-3X9P                                                                      AACGCCATTT TCAGTGTTAC A C                                                                              [SEQ 12]                                             EXON 10                                                                       P53-5X10P                                                                     TCATCCCTCA TAAACTCAAA CAA                                                                              [SEQ 13]                                             P53-3X10P                                                                     CTCGACGCAA CAATCTCCTT A  [SEQ 14]                                             EXON 11                                                                       P53-5X11P                                                                     GCCACAGACC CTCTCACTCA T  [SEQ 15]                                             P53-3X11P                                                                     TGCTTCTGAC GCACACCTAT T  [SEQ 16]                                             ______________________________________                                    

These primers result in amplified products with normal fragment lengthsof 331 bp for exon 1, 162 bp for exon 3, 382 bp for exon 4, 268 bp forexon 5, 247 bp for exon 6, 209 bp for exon 9, 390 bp for exon 10, and256 bp for exon 11. The control sequence produces a further fragmenthaving a length which should not correspond to any of the expectedlengths.

In multiplex pool B, exons 2 and 8 are amplified (along with a controlsequence). The members of this pool are selected because they all use ahybridization temperature of 62° C., and none of the expected fragmentlengths will overlap in an electrophoresis gel. One of each pair ofprimers is labeled at the 5 prime end with an identifiable marker suchas fluorescein, rhodamine or cyanine. The primers are:

    ______________________________________                                        EXON 2                                                                        P53-5X2P                                                                      ACCCAGGGTT GGAAGCGTCT    [SEQ 17]                                             P53-3X2P                                                                      GACAAGAGCA GAAAGTCAGT CC [SEQ 181                                             EXON 8                                                                        P53-5X8P                                                                      GACAAGGGTG GTTGGGAGTA GATG                                                                             [SEQ 19]                                             P53-3X8P                                                                      GCAAGGAAAG GTGATAAAAG TGAA                                                                             [SEQ 20]                                             ______________________________________                                    

These primers result in amplified products with normal fragment lengthsof 261 bp for exon 2 and 320 bp for exon 8. The control sequenceproduces a further fragment having a length which should not correspondto any of the expected lengths.

Finally, in multiplex pool C, only exon 7 is amplified (along with acontrol sequence). The primers for exon 7 require a hybridizationtemperature of 64° C. unlike any of the other amplification primers. Oneof the pair of primers is labeled at the 5 prime end with anidentifiable marker such as fluorescein, rhodamine or cyanine. Theprimers are:

    ______________________________________                                        EXON 7                                                                        P53-5X7P                                                                      GGCGACAGAG CGAGATTCCA   [SEQ 21]                                              P53-3X7P                                                                      GGGTCAGCGG CAAGCAGAGG   [SEQ 22]                                              ______________________________________                                    

These primers result in an amplified product with a normal fragmentlength of 286 bp. The control sequence produces a further fragmenthaving a length which should not correspond to this expected length.

After amplification, the products from each amplification reaction aredenatured and loaded into a polyacrylamide gel for electrophoreticseparation. In the preferred embodiment, electrophoretic separationtakes place in a semi-automated electrophoresis apparatus such as in aPharmacia A.L.F.™ automated sequencer. In another embodiment,electrophoretic separation takes place in a microgel disclosed in U.S.patent application Ser. No. 08/332,577, which is incorporated herein byreference. In either embodiment, the amplification products migratethrough the gel at a rate determined by their length and are detectedusing the fluorescence of the fluorescent molecule (either fluorescein,rhodamine or cyanine) which was attached to the primers.

The products of each amplification set are separated in a different laneof the gel, unless molecules which fluoresce at different wavelengthshave been used as labels on the primers, in which case the products maybe run in the same lane, and distinguished by wavelength of fluorescenceemission. The fragment sizes and amounts are compared to the expectedsizes of the normal gene fragments. If length mutation is detected, thenthe sample is concluded to contain a mutation in the p53 gene. If theamount of amplified fragment is 25% or more below or above the amount ofthe wild type fragment (using the amount of control fragment as astandard of comparison), then the sample is concluded to contain an LOH(loss of heterozygosity) or gene amplification mutation, respectively. Apatient report was prepared for those samples that are identified ashaving a fragment length or quantity mutation. Where no length orquantity mutation is detected, then the sample was re-examined using thenext and final level of the hierarchy.

Level 3 DNA Sequence Analysis

The group of patient samples that have proven negative for p53 mutationsunder the protein immunoassay and the fragment length/quantity analysismay be re-examined for point mutations in the DNA sequence.

DNA from an individual patient is purified as in the fragmentlength/quantity analysis, above. Exon containing fragments of the p53gene are amplified using primers and conditions listed in Table 1. Theprimers are the same as those used in the fragment length/quantityanalysis. However, in some cases the primers are used in differentcombinations. For example, because exons 5 and 6 lie in reasonably closeproximity on genomic DNA, it is adequate for amplification to use theprimer at the 5 prime end of exon 5 and at the 3 prime end of exon 6,and to amplify both exons together on a single fragment. The resultingamplified fragment is suitable for sequencing either exon 5 or exon 6.The same situation is found with exons 3 and 4.

                                      TABLE 1                                     __________________________________________________________________________    PRE-SEQUENCING AMPLIFICATION CONDITIONS                                                         initial                        final                                          denaturing                                                                          denaturing                                                                           anneal Extension  extension                    EXON                                                                              5' primer                                                                            3' primer                                                                            temp/time                                                                           temp/time                                                                            temp/time                                                                            temp/ time                                                                           cycles                                                                            temp/time                    __________________________________________________________________________    1   p53-5X1PCR                                                                           p53-3X1PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min                                                           72° C./3 min          2   p53-5X2PCR                                                                           p53-3X2PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 62° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          3   p53-5X3PCR                                                                           p53-3X3PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          4   p53-5X4PCR                                                                           p53-3X4PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          5   p53-5X5PCR                                                                           p53-3X5PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          6   p53-5X6PCR                                                                           p53-3X6PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          7   p53-5X7PCR                                                                           p53-3X7PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 64° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          8   p53-5X8PCR                                                                           p53-3X8PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 62° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          9   p53-5X9PCR                                                                           p53-3X9PCR                                                                           94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          10  p53-5X10PCR                                                                          p53-3X10PCR                                                                          94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3 min          11  p53-5X11PCR                                                                          p53-3X10PCR                                                                          94° C./4 min                                                                 94° C./30 min                                                                 60° C./50 min                                                                 70° C./60 min                                                                 30  72° C./3              __________________________________________________________________________                                                     min                      

Once the sets of exons are amplified, DNA sequencing reactions may beperformed on the amplified sample. Dideoxy sequencing primers have beendeveloped for both strands of each exon (except exon 3 which has onlyone sequencing primer) of the p53 gene, and are listed below:

    ______________________________________                                        EXON 1                                                                        P53-5X1SEQ CCCATTACTT CCCCTTACTT CTCA*                                                                   [SEQ 1]                                            P53-3X1SEQ CCCCACCCCC ACCCATTTT                                                                          [SEQ 2]                                            EXON 2                                                                        P53-5X2SEQ CCACCCTTCC AACCCTCTC*                                                                         [SEQ 23]                                           P53-3X2SEQ GCTACCCCGC TCGGCTTGC                                                                          [SEQ 24]                                           EXON 3                                                                        P53-3X3SEQ ATGGCTCAAA ACACCACT*                                                                          [SEQ 25]                                           EXON 4                                                                        P53-5X4SEQ CGGCCTGAGG ACCTGCTC                                                                           [SEQ 26]                                           P53-3X4SEQ ATACCCCCAC GCATTCAA                                                                           [SEQ 27]                                           EXON 5                                                                        PS3-5X5SEQ CACTTCTCCC CTGACTTT*                                                                          [SEQ 28]                                           P53-3X5SEQ CCTCGGGACC CTCCGCAA                                                                           [SEQ 29]                                           EXON 6                                                                        P53-5X6SEQ TGGTTGCCCA CCCTCCCC*                                                                          [SEQ 30]                                           P53-3X6SEQ CCACTGACAA CCACCC                                                                             [SEQ 31]                                           EXON 7                                                                        P53-5X7SEQ CTCCCCTGCT TCCCACA*                                                                           [SEQ 32]                                           P53-3X7SEQ TCACCCCCAA CCACACC                                                                            [SEQ 33]                                           EXON 8                                                                        P53-5X8SEQ ATGCCACACG TACCACC*                                                                           [SEQ 34]                                           P53-3X8SEQ CATAACTGCA CCCTTCG                                                                            [SEQ 35]                                           EXON 9                                                                        P53-5X9SEQ GGAGGAGACC AAGGGTGC                                                                           [SEQ 36]                                           P53-3X9SEQ GGAAACTTTC CACTTGA*                                                                           [SEQ 37]                                           EXON 10                                                                       P53-5X10SEQ CCATCTTTTA ACTCAGGT*                                                                         [SEQ 38]                                           P53-3X10SEQ CATGAAGGCA GGATGAG                                                                           [SEQ 39]                                           EXON 11                                                                       P53-5X11SEQ AGACCCTCTC ACTCATG                                                                           [SEQ 40]                                           P53-3X11SEQ CAAGCAAGGG TTCAAAG*                                                                          [SEQ 41]                                           ______________________________________                                    

The primers are generally nested inside the amplification primers, i.e.closer to the exon, although in some cases the preferred sequencingprimer is in fact the amplification primer. The 5 prime sequencingprimer provides the sequence from the sense strand; the 3 primesequencing primer provides the sequence from the anti-sense strand ofthe p53 gene. Only one of these primers needs to be used to obtainsequence from the exon in question. The preferred primer is marked withan asterisk in the list above. The preferred primer for sequencing isconjugated to a fluorescent molecule such as fluorescein, rhodamine orcyanine for detection, although other forms of detectable labels,including labeled nucleotides or dideoxynucleotides may be employed.

Dideoxy DNA sequencing is performed using the well known method ofSanger et al., "DNA sequencing with chain terminating inhibitors", ProcNatl Acad Sci USA 74:5463-5467 (1977), as modified for use withSequenase™ Version 2.0 (United States Biochemical Corporation, ClevelandOhio) Products of the DNA sequencing reaction are analyzed using asemi-automated electrophoresis apparatus as in the DNA fragmentlength/quantity analysis described above.

Current epidemiological data was used to determine which gene fragmentsare preferably sequenced first. Mutations have been detected in allexons, but are extremely rare in exon 1 and in the 3'-end of exon 11. Itis therefore preferable to sequence exons 2-10 ahead of exons 1 and 11.Currently the preferred order of sequencing begins with exon 6, then 7,then 5. The remaining exons are sequenced in turn.

Samples wherein mutations are detected relative to the wild-type p53gene are recorded and reported to the individual patient's file. Whereno mutation is identified, another exon containing fragment of theindividual sample is sequenced. Again mutations are identified andreported. If, after sequencing all the exon containing fragments of thegene, there are no mutations identified, it is concluded that theindividual sample contains no p53 mutation.

All final results of testing are reported to the patient file. Thereport is communicated to the patient by electronic transmission orwritten report, or both.

EXAMPLE 2

A second embodiment of the p53 assay skips the protein immunoassay leveland begins with the preparation of DNA from a patient blood sample.Genomic DNA is prepared from a blood sample as in Example 1 and it isassayed according to the DNA measurement procedures of Example 1,including fragment length/quantity analysis and DNA sequence analysis.Patient reports are prepared when diagnosis of the presence or absenceof p53 mutation is determined.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 41                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 1 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                            #                24ACTT GTCA                                                  - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 1 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                            # 19               TTT                                                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 3 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                            # 20               TGCT                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 3 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                            # 19               TGT                                                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 4 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                            #                24TCTT TTCA                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 4 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                            #                 22TAC GG                                                    - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 5 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                            # 20               GACT                                                       - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 5 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                            # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 6 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                            # 20               GACA                                                       - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 6 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                           # 19               CCA                                                        - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 9 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                           # 19               AGG                                                        - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 9 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                           #                 22TAG AC                                                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 10 of human p53 gene              -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                           #                23CAAA CAA                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 10 of human p53 gene              -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                           #21                GGTT A                                                     - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 11 of human p53 gene              -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                           #21                CTCA T                                                     - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 11 of human p53 gene              -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                           #21                CTAT T                                                     - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 2 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                           # 20               GTCT                                                       - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 2 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                           #                 22AGT CC                                                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 8 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                           #                24AGTA GATG                                                  - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 8 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                           #                24AAAG TGAA                                                  - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 7 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                           # 20               TCCA                                                       - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: primer fo - #r exon 7 of human p53 gene               -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                           # 20               GAGG                                                       - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 2 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                           # 19               CTC                                                        - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 2 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                           # 19               TGG                                                        - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 3 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                           #  18              GT                                                         - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 4 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                           #  18              TC                                                         - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 4 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                           #  18              AA                                                         - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 5 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                           #  18              TT                                                         - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 5 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                           #  18              AA                                                         - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 6 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                           #  18              CC                                                         - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 6 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                           #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 7 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                           #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 7 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                           #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 8 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                           #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 8 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                           #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 9 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                           #  18              GG TGC                                                     - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 9 of human p53 gene                                          -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                           #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 10 of human p53 gene                                         -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                           #  18              GT                                                         - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 10 of human p53 gene                                         -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                           #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: yes                                                    -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 11 of human p53 gene                                         -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                           #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: genomic DNA                                         -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                           #primer for exon 11 of human p53 gene                                         -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                           #   17             G                                                          __________________________________________________________________________

We claim:
 1. A kit for the identification of mutations in the p53 gene,comprising at least one primer pair for amplification of an exon of thep53 gene, each member of said primer pair being labeled with adetectable label, wherein the kit comprises a primer pair selected fromthe group consisting of:

    (a)  CGGATTACTT GCCCTTACTT GTCA                                                                       [SEQ 1]                                               and                                                                                CCCCAGCCCC AGCGATTTT                                                                            [SEQ 2];                                               (b)  CATGGGACTG ACTTTCTGCT                                                                           [SEQ 3]                                                and                                                                                GGACGGCAAG GGGGACTGT                                                                            [SEQ 4];                                               (c) CTGGTCCTCT GACTGCTCTT TTCA                                                                       [SEQ 5]                                                and                                                                                AAAGAAATGC AGGGGGATAC GG                                                                        [SEQ 6];                                               (d) TGTTCACTTG TGCCCTGACT                                                                            [SEQ 7]                                                and                                                                                CAGCCCTGTC GTCTCTCCAG                                                                           [SEQ 8];                                               (e) CTGGGGCTGG AGAGACGACA                                                                            [SEQ 9]                                                and                                                                                GGAGGGCCAC TGACAACCA                                                                            [SEQ 10];                                              (f) GCGGTGGAGG AGACCAAGG                                                                             [SEQ 11]                                               and                                                                                AACGGCATTT TGAGTGTTAG A C                                                                       [SEQ 12]:                                              (g)  TGATCCGTCA TAAAGTCAAA CAA                                                                       [SEQ 13]                                               and                                                                                GTGGAGGCAA GAATGTGGTT A                                                                         [SEQ 14];                                              (h) GGCACAGACC CTCTCACTCA T                                                                          [SEQ 15]                                               and                                                                                TGCTTCTGAC GCACACCTAT T                                                                         [SEQ 16];                                              (i)  ACCCAGGGTT GAAGCGTCT                                                                            [SEQ 17]                                               and                                                                                GACAAGAGCA GAAAGTCAGT CC                                                                        [SEQ 18];                                              (j)  GACAAGGGTG GTTGGGAGTA GATG                                                                      [SEQ 19]                                               and                                                                                GCAAGGAAAG GTGATAAAAG TGAA                                                                      [SEQ 20]; and                                          (k)  GGCGACAGAG CGAGATTCCA                                                                           [SEQ 21]                                               and                                                                                GGGTCAGCGG CAAGCAGAGG                                                                           [SEQ 22].                                          


2. A kit according to claim 1, further comprising at least one p53sequencing primer for the exon.
 3. A kit according to claim 2, whereinthe sequencing primer is selected from the group consisting of

    ______________________________________                                        CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1],                                              CCCCAGCCCC AGCGATTTT    [SEQ 2],                                              CCAGGGTTGG AAGCGTCTC    [SEQ 23],                                             GCTAGGGGGC TGGGGTTGG    [SEQ 24],                                             ATGGGTGAAA AGAGCAGT     [SEQ 25],                                             GGGGCTGAGG ACCTGGTC     [SEQ 261,                                             ATACGGCCAG GCATTGAA     [SEQ 27],                                             CACTTGTGCC CTGACTTT     [SEQ 28]                                              CCTGGGGACC CTGGGCAA     [SEQ 29],                                             TGGTTGCCCA GGGTCCCC     [SEQ 30],                                             CCACTGACAA CCACCC       [SEQ 31],                                             CTCCCCTGCT TGCCACA      [SEQ 321,                                             TCAGCGGCAA GCAGAGG      [SEQ 33],                                             ATGGGACAGG TAGGACC      [SEQ 34],                                             CATAACTGCA CCCTTGG      [SEQ 35],                                             GGAGGAGACC AAGGGTGC     [SEQ 36],                                             GGAAACTTTC CACTTGA      [SEQ 37],                                             CCATCTTTTA ACTCAGGT     [SEQ 38],                                             CATGAAGGCA GGATGAG      [SEQ 39],                                             AGACCCTCTC ACTCATG      [SEQ 40], and                                         CAAGCAAGGG TTCAAAG      [SEQ 41].                                             ______________________________________                                    


4. A kit according to claim 1, wherein the kit comprises at least oneprimer cocktail containing a mixture of primers effective to coamplify aplurality of exons of the p53 gene.
 5. A kit according to claim 4,wherein the primer cocktail contains a mixture of primers effective toamplify exons 2 and 8 of the p53 gene.
 6. A kit according to claim 5,wherein the primer cocktail comprises primers of the sequence:

    ______________________________________                                        ACCCAGGGTT GGAAGCGTCT   [SEQ 17]                                              GACAAGAGCA GAAAGTCAGT CC                                                                              [SEQ 18]                                              GACAAGGGTG GTTGGGAGTA GATG                                                                            [SEQ 19] and                                          GCAAGGAAAG GTGATAAAAG TGAA                                                                            [SEQ 20].                                             ______________________________________                                    


7. A kit according to claim 5, wherein the primer cocktail contains amixture of primers effective to amplify exons 1, 3, 4, 5, 6, 9 and 10 ofthe p53 gene.
 8. A kit according to claim 7, wherein the primer cocktailcomprises primers of the sequence:

    ______________________________________                                        CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1]                                               CCCCAGCCCC AGCGATTTT    [SEQ 2]                                               CATGGGACTG ACTTTCTGCT   [SEQ 3]                                               GGACGGCAAG GGGGACTGT    [SEQ 4]                                               CTGGTCCTCT GACTGCTCTT TTCA                                                                            [SEQ 5]                                               AAAGAAATGC AGGGGGATAC GG                                                                              [SEQ 6]                                               TGTTCACTTG TGCCCTGACT   [SEQ 7]                                               CAGCCCTGTC GTCTCTCCAG   [SEQ 8]                                               CTGGGGCTGG AGAGACGACA   [SEQ 9]                                               GGAGGGCCAC TGACAACCA    [SEQ 10]                                              GCGGTGGAGG AGACCAAGG    [SEQ 11]                                              AACGGCATTT TGAGTGTTAG AC                                                                              [SEQ 12]                                              TGATCCGTCA TAAAGTCAAA CAA                                                                             [SEQ 13]                                              GTGGAGGCAA GAATGTdGTT A [SEQ 14]                                              GGCACAGACC CTCTCACTCA T [SEQ 15] and                                          TGCTTCTGAC GCACACCTAT T [SEQ 16].                                             ______________________________________                                    

to produce a reaction mixture and thermally cycling the reaction mixtureto produce at least one species of amplified product.
 9. A primercocktail comprising a plurality of oligonucleotide primer pairs foramplification of exons of the p53 gene, including a first primer pairfor amplification of only exon 2 and a second primer pair foramplification of only exon 8 of the p53 gene.
 10. A primer cocktailcomprising a plurality of oligonucleotide primers for amplification ofexons 2 and 8 of the p53 gene, wherein the primer cocktail comprisesprimers of the sequence:

    ______________________________________                                        ACCCAGGGTT GGAAGCGTCT   [SEQ 17]                                              GACAAGAGCA GAAAGTCAGT CC                                                                              [SEQ 18]                                              GACAAGGGTG GTTGGGAGTA GATG                                                                            [SEQ 19] and                                          GCAAGGAAAG GTGATAAAAG TGAA                                                                            [SEQ 20].                                             ______________________________________                                    


11. A primer cocktail comprising a plurality of oligonucleotide primerpairs for amplification of exons of the p53 gene, including separateprimer pairs for amplification of each of exons 1, 3, 4, 5, 6, 9 and 10of the p53 gene.
 12. A primer cocktail comprising a plurality ofoligonucleotide primers for amplification of exons 1, 3, 4, 5, 6, 9 and10 of the p53 gene, wherein the primer cocktail comprises primers of thesequence:

    ______________________________________                                        CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1]                                               CCCCAGCCCC AGCGATTTT    [SEQ 2]                                               CATGGGACTG ACTTTCTGCT   [SEQ 3]                                               GGACGGCAAG GGGGACTGT    [SEQ 4]                                               CTGGTCCTCT GACTGCTCTT TTCA                                                                            [SEQ 5]                                               AAAGAAATGC AGGGGGATAC GG                                                                              [SEQ 6]                                               TGTTCACTTG TGCCCTGACT   [SEQ 7]                                               CAGCCCTGTC GTCTCTCCAG   [SEQ 8]                                               CTGGGGCTGG AGAGACGACA   [SEQ 9]                                               GGAGGGCCAC TGACAACCA    [SEQ 10]                                              GCGGTGGAqG AGACCAAGG    [SEQ 11]                                              AACGGCATTT TGAGTGTTAG AC                                                                              [SEQ 12]                                              TGATCCGTCA TAAAGTCAAA CAA                                                                             [SEQ 13]                                              GTGGAGGCAA GAATGTGGTT A [SEQ 14]                                              GGCACAGACC CTCTCACTCA T [SEQ 15] and                                          TGCTTCTGAC GCACACCTAT T [SEQ 16].                                             ______________________________________                                    


13. A kit according to claim 2, wherein the kit comprises at least oneprimer cocktail containing a mixture of primers effective to coamplify aplurality of exons of the p53 gene.
 14. A kit according to claim 3,wherein the kit comprises at least one primer cocktail containing amixture of primers effective to coamplify a plurality of exons of thep53 gene.
 15. A method for amplification of at least one exon of thehuman p53 gene in a sample comprising the steps of combining the samplewith at least one primer pair selected from the group consisting of:

    ______________________________________                                        (a)   CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1]                                         and                                                                                 CCCCAGCCCC AGCGATTTT    [SEQ 2];                                        (b)   CATGGGACTG ACTTTCTGCT   [SEQ 3]                                         and                                                                                 GGACGGCAAG GGGGACTGT    [SEQ 4];                                        (c)   CTGGTCCTCT GACTGCTCTT TTCA                                                                            [SEQ 5]                                         and                                                                                 AAAGAAATGC AGGGGGATAC GG                                                                              [SEQ 6];                                        (d)   TGTTCACTTG TGCCCTGACT   [SEQ 7]                                         and                                                                                 CAGCCCTGTC GTCTCTCCAG   [SEQ 8];                                        (e)   CTGGGGCTGG AGAGACGACA   [SEQ 9]                                         and                                                                                 GGAGGGCCAC TGACAACCA    [SEQ 10];                                       (f)   GCGGTGGAGG AGACCAAGG    [SEQ 11]                                        and                                                                                 AACGGCATTT TGAGTGTTAG AC                                                                              [SEQ 12];                                       (g)   TGATCCGTCA TAAAGTCAAA CAA                                                                             [SEQ 13]                                        and                                                                                 GTGGAGGCAA GAATGTGGTT A [SEQ 14];                                       (h)   GGCACAGACC CTCTCACTCA T [SEQ 15]                                        and                                                                                 TGCTTCTGAC GCACACCTAT T [SEQ 16];                                       (i)   ACCCAGGGTT GGAAGCGTCT   [SEQ 17]                                        and                                                                                 GACAAGAGCA GAAAGTCAGT CC                                                                              [SEQ 18];                                       (j)   GACAAGGGTG GTTGGGAGTA GATG                                                                            [SEQ 19]                                        and                                                                                 GCAAGGAAAG GTGATAAAAG TGAA                                                                            [SEQ 20]; and                                   (k)   GGCGACAGAG CGAGATTCCA   [SEQ 21]                                        and                                                                                 GGGTCAGCGG CAAGCAGAGG   [SEQ 22];                                       ______________________________________                                    


16. The method according to claim 15, wherein sample is combined with atleast one primer cocktail containing a mixture of primers effective tocoamplify a plurality of exons of the p53 gene.
 17. The method accordingto claim 16, wherein the primer cocktail contains a mixture of primerseffective to amplify exons 2 and 8 of the p53 gene.
 18. The methodaccording to claim 17, wherein the primer cocktail comprises primers ofthe sequence:

    ______________________________________                                        ACCCAGGGTT GGAAGCGTCT   [SEQ 17]                                              GACAAGAGCA GAAAGTCAGT CC                                                                              [SEQ 18]                                              GACAAGGGTG GTTGGGAGTA GATG                                                                            [SEQ 19] and                                          GCAAGGAAAG GTGATAAAAG TGAA                                                                            [SEQ 20].                                             ______________________________________                                    


19. The method according to claim 16, wherein the primer cocktailcontains a mixture of primers effective to amplify exons 1, 3, 4, 5, 6,9 and 10 of the p53 gene.
 20. The method according to claim 19, whereinthe primer cocktail comprises primers of the sequence:

    ______________________________________                                        CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1]                                               CCCCAGCCCC AGCGATTTT    [SEQ 2]                                               CATGGGACTG ACTTTCTGCT   [SEQ 3]                                               GGACGGCAAG GGGGACTGT    [SEQ 4]                                               CTGGTCCTCT GACTGCTCTT TTCA                                                                            [SEQ 5]                                               AAAGAAATGC AGGGGGATAC GG                                                                              [SEQ 6]                                               TGTTCACTTG TGCCCTGACT   [SEQ 7]                                               CAGCCCTGTC GTCTCTCCAG   [SEQ 8]                                               CTGGGGCTGG AGAGACGACA   [SEQ 9]                                               GGAGGGCCAC TGACAACCA    [SEQ 10]                                              GCGGTGGAGG AGACCAAGG    [SEQ 11]                                              AACGGCATTT TGAGTGTTAG AC                                                                              [SEQ 12]                                              TGATCCGTCA TAAAGTCAAA CAA                                                                             [SEQ 13]                                              GTGGAGGCAA GAATGTGGTT A [SEQ 14]                                              GGCACAGACC CTCTCACTCA T [SEQ 15] and                                          TGCTTCTGAC GCACACCTAT T [SEQ 16].                                             ______________________________________                                    


21. A method for sequencing a portion of a human p53 gene in a samplecomprising the steps of combining the sample with a sequencing reactionmixture containing a sequencing primer selected from the groupconsisting of

    ______________________________________                                        CGGATTACTT GCCCTTACTT GTCA                                                                            [SEQ 1],                                              CCCCAGCCCC AGCGATTTT    [SEQ 2],                                              CCAGGGTTGG AAGCGTCTC    [SEQ 23],                                             GCTAGGGGGC TGGGGTTGG    [SEQ 24],                                             ATGGGTGAAA AGAGCAGT     [SEQ 25],                                             GGGGCTGAGG ACCTGGTC     [SEQ 26]                                              ATACGGCCAG GCATTGAA     [SEQ 27],                                             CACTTGTGCC CTGACTTT     [SEQ 28],                                             CCTGGGGACC CTGGGCAA     [SEQ 29],                                             TGGTTGCCCA GGGTCCCC     [SEQ 30],                                             CCACTGACAA CCACCC       [SEQ 31],                                             CTCCCCTGCT TGCCACA      [SEQ 32],                                             TCAGCGGCAA GCAGAGG      [SEQ 33],                                             ATGGGACAGG TAGGACC      [SEQ 34],                                             CATAACTGCA CCCTTGG      [SEQ 35],                                             GGAGGAGACC AAGGGTGC     [SEQ 36],                                             GGAAACTTTC CACTTGA      [SEQ 37],                                             CCATCTTTTA ACTCAGGT     [SEQ 38],                                             CATGAAGGCA GGATGAG      [SEQ 39],                                             AGACCCTCTC ACTCATG      [SEQ 40], and                                         CAAGCAAGGG TTCAAAG      [SEQ 41];                                             ______________________________________                                    

producing sequencing fragments by extension of the sequencing primer;and evaluating the length of the sequencing fragments to determine thesequence.